Synthesis of conjugated octadecatrienoic acid using enzymes from tung

ABSTRACT

Conjugated trienoic fatty acid was formed in a mixture containing CoASH, NADH, either ADP or ATP, MgSO4, castor fatty acid, and enzyme prepared from tung nuts. The enzyme was prepared as a protein, which is soluble in 0.1 M tris- HCl buffer (pH 7.2), from acetone-insoluble powder of maturing tung nuts.

O Umted States Patent [151 3,661,71 0

Jacks et al. 1 May 9, 1972 [54] SYNTHESIS OF CONJUGATED References Cited OCTADECATRIENOIC ACID USING OTHER PUBLICATIONS ENZYNIES FROM TUNG Markley, K. S., Fatty Acids," Part 3, p 1509- 23 [72] Inventors: Thomas J. Jacks, Metajrie; Lawrence Y. Publishers-Interscience,1964.

Yatsu, New Orleans, both of La. [73] Assignee: The United States of America as Pmfmry Lou's Menace" represented by the Secretary of Agricub Assistant Examiner-Gary M. Nath mm Attorney-R. Hoffman and W. Bler [2]] Appl' 39921 Conjugated trienoic fatty acid was formed in a mixture containing CoASH, NADH, either ADP or ATP, MgSO castor [52] US. Cl ..l95/30, 195/66 fatty acid and enzyme prepared from tung nuts. The enzyme "E (1 1/00 was prepared as a protein, which is soluble in 0.1 M tris- HCl [58] Field Of Search ..l95/30, 66, 51 buffer (PH 7.2), from acetonednsoluble powder of maturing tung nuts.

7 Claims, No Drawings SYNTHESIS OF CONJUGATED OCTADECATRIENOIC ACID USING ENZYMES FROM TUNG A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to The Government of the United States of America.

This invention relates to the synthesis of conjugated trienoic fatty acids. More specifically, this invention relates to the synthesis of conjugated trienoic fatty acid in vitro as catalyzed by enzymes prepared from developing tung nuts and in the presence of certain enzymic cofactors.

The main object of this invention is the synthesis of conjugated trienoic fatty acids. More specifically, the object is the synthesis of conjugated octadecatrienoic acid. Lesser objects are the use of enzymes as the catalytic agents for this synthesis and the preparation of such enzymes from a plant source which, in this case, is tung (Aleurites fordii, Hemsl).

Conjugated trienoic fatty acids are in great demand as drying oils in paint and other coatings. These acids are extracted from plant materials, such as tung nuts, but have never been synthesized in vitro. The object ofthis invention is the synthesis of conjugated trienoic fatty acid in vitro. Synthesis of conjugated trienoic fatty acids in vitro provides a steady supply of such acids.

We have now found that acetone-insoluble powders prepared from developing tung nuts catalyze the formation of conjugated octadecatrienoic acid from fatty acids of castor oil. The preferred synthesis required reduced coenzyme A (CoASH reduced nicotinamide-adenine dinucleotide (NADH), enzyme from tung nuts, and either adenosine-5'- diphosphate (ADP) or adenosine-S'-triphosphate (ATP) for best results.

Developing tung nuts were collected before, during, and after the period of active oil accumulation in the endosperm (L. Y. Yatsu and M. R. Easterling, Plant Physiol. 3911017, 1964). Thinly sliced tissue was macerated in liquid nitrogen with a mortar and pestle. The small, frozen pieces were added to an equal weight of sand and homogenized at 78 C with sufficient acetone to ensure fluidity of the homogenate. The insoluble residue was washed by vacuum filtration with acetone at 78 C until the filtrate appeared colorless and then with sufficient ether to displace residual acetone. Ether was removed by immediately fluffing the powder at room temperature in an exhaust hood. The powder was further dried over P in a vacuum of 0.05 torr and then stored in sealed ampules at 20 C.

Enzyme was prepared by triturating the powder in 0.1 M tris-HCl buffer (pl-l 7.2), filtering the suspension through glass wool, and centrifuging the filtrate at 1,100 g for min. Dithiothreitol was added to the supernatant to a concentration of 4 l0 M. This enzyme preparation, containing to 35 mg of protein per ml (mostly storage protein of the seed), was homogenized with other components of the reaction mixture and the mixture allowed to incubate at 30 C with continual homogenization. At various time intervals portions of 0.6 ml were removed and added to 0.4 m1 of 0.5 N HCl Fatty acids were extracted from each acidified reaction mixture into 3.0 ml of hexane (spectro-photometric grade) and the content of conjugated trienoic fatty acid was determined spectrophotometrically (A.O.C.S. Official Method Cd 7-58, Revised 1959; J. S. Hoffman, R. T. OConnor, D. C. Heinzelman, and W. G. Bickford, J. Am. Oil Chem. Soc. 34:338, 1957).

Best results were obtained when the reaction mixture contained, in 0.6 ml or 0.8 ml, 5 umoles of CoASl-l, l1 pmoles of NADH, l0 umoles of ATP or ADP, 1.2 umoles of MgSO.,, 230 [1.1110165 of fatty acid from castor oil, and sufficient 0.5 N KOl-l to neutralize the above mixture. The addition of 1.4 or 1.2 ml of enzyme preparation to this mixture obtained synthesis of conjugated octadecatrienoic fatty acid. Only enzyme preparations from acetone-insoluble powders of maturing tung nuts that were rapidly accumulating oil were enzymically active. Neither immature nuts that contained small amounts of endosperm nor fully mature, quiescent nuts provided active powders EXAMPLE 1 Developing tung nuts were collected before, during, and after the period of active oil accumulation in the endosperm (L. Y. Yatsu and M. R. Easterling, Plant Physiol. 39: 1017 1964). Thinly sliced tissue was macerated in liquid nitrogen with a mortar and pestle. The small, frozen pieces were added to an equal weight of sand and homogenized at -78 C with sufficient acetone to ensure fluidity of the homogenate. The insoluble residue was washed by vacuum filtration with acetone at 78 C until the filtrate appeared colorless and then with sufficient ether to displace residual acetone. Ether was removed by immediately fluffing the powder at room temperature in an exhaust hood. The powder was further dried over P 0 in a vacuum of 0.05 torr and then stored in sealed ampules at -20 C.

The enzyme was prepared by triturating the powder in 0.1 M tris-HC] buffer pH 7.2), filtering the suspension through glass wool, and centrifuging the filtrate at 1,100 g for 10 min. Dithiothreitol was added to the supernatant to a concentration of 4X10"M. This enzyme preparation contains 15 to 35 mg of protein per ml.

The enzyme preparation containing 49 mg of protein, pmoles of tris-HC! buffer (pl-1 7.2) and 4.8 pmoles of dithiothreitol was homogenized with Spmoles of CoASH, ll umoles of NADH, l0 umoles of ATP, 1.2 umoles of MgSO,, 230 umoles of castor oil fatty acid, sufficient 0.5 N KOH to neutralize the mixture. The mixture was allowed to incubate for 45 minutes at 30 C with continual homogenization At various time intervals portions of 0.6 ml were removed and added to 0.4 ml of 0.5 N HCl. Fatty acids were extracted from each acidified reaction mixture into 3.0 ml of hexane (spectrophotometric grade) and the content of conjugated trienoic fatty acid was determined spectrophotometrically (A.O.C.S. Official Method Cd 7-58, Revised 1959; .1. S. Hoffman, R. T. OConnor, D. C. Heinzelman, and W. G. Bickford, J. Am. Oil Chem. Soc. 34: 338, 1957).

EXAMPLE 2 The reaction given in Example 1 was repeated except that CoASH was omitted. After 45 min. 12.4 my. moles of conjugated trienoic fatty acid was produced.

EXAMPLE 3 The reaction given in Example 1 was repeated except that NADH was omitted. After 45 min. 12.3 mp. moles of conjugated trienoic fatty acid was produced.

EXAMPLE 4 The reaction given in Example 1 was repeated except that NADH was omitted and l l pmoles of oxidized nicotinamideadenine dinucleotide, NAD, was present. After 45 min. 29.9 mu moles of conjugated trienoic fatty acid was produced.

EXAMPLE 5 The reaction given in Example 1 was repeated except that NADH was absent and l l umoles of oxidized nicotinamideadenine dinucleotide phosphate, NADP, was present. After 45 min. 9.2 m,u. moles of conjugated trienoic fatty acid was produced.

EXAMPLE 6 The reaction given in Example 1 was repeated except that ATP was omitted. After 45 min. 1.5 mg moles of conjugated trienoic fatty acid was produced.

EXAMPLE 7 The reaction given in Example 1 was repeated except that ATP was omitted and 10 umoles of ADP were present. After 45 min. 31.0 moles of conjugated trienoic fatty acid was produced.

We claim:

1. A process for preparing an enzyme, useful in the synthesis of conjugated trienoic fatty acid, which process comprises:

a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil,

b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about 78 C,

c. washing the acetone-insoluble residue with acetone at a temperature of about 78 C and then with ether,

d. removing the ether from (c) and drying the powder in vacuo,

e. triturating the powder from (d) in 0.1 M tris-HCl buffer,

f. filtering the suspension from (e) through glass wool,

g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes,

h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-l-lCl buffer. 2. A process for synthesizing conjugated trienoic acid in vitro which process comprises:

a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil,

b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about 78 C,

c. washing the acetone-insoluble residue with acetone at a temperature of about 78 C and then with ether,

d. removing the ether from (c) and drying the powder in vacuo,

e. triturating the powder from (d) in 0.1 M tris-HC] buffer,

f. filtering the suspension from (e) through glass wool,

g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes,

h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HC] buffer, i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 15.7 mM of reduced nicotinamide-adenine dinucleotide about 11.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-S '-diphosphate and adenosinetriphosphate, about 1.7 mM MgSO,, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOH to neutralize the mixture, incubating the reaction mixture from (i) at 30 C for a period of about 45 minutes, it. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HC] and then about parts of hexane. 3. A process for synthesizing conjugated trienoic acid in vitro which process comprises:

a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil,

b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about 78 C,

c. washing the acetone-insoluble residue with acetone at a temperature of about 78 C and then with ether,

d. removing the ether from (c) and drying the powder in vacuo, I

e. triturating the powder from (d) in 0.1 M tris-HCl buffer,

f. filtering the suspension from (e) through glass wool,

g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes,

h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer,

i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 066 parts of a mixture consisting of about 15.7 mM of reduced nicotinamide-adenine dinucleotide, about 11.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5'-diphosphate and adenosine-S'-triphosphate, about 1.7 mM M about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOH to neutralize the mixture,

j. incubating the reaction mixture from (i) at 30 C for a period of about 45 minutes,

k. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.

4. A process for synthesizing conjugated trienoic acid in vitro which process comprises:

a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil,

b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about 78 C,

c. washing the acetone-insoluble residue with acetone at a temperature of about 78 C and then with ether,

d. removing the ether from (c) and drying the powder in vacuo,

e. triturating the powder from (d) in 0.1 M tris-HCl buffer,

f. filtering the suspension from (e) through glass wool,

g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes,

h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer,

. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 1 1.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5'-diphosphate and adenosine-5- triphosphate, about 1.7 mM MgSO,, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOl-l to neutralize the mixture,

j. incubating the reaction mixture from (i) at 30 C for a period of about 45 minutes,

k. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.

5. A process for synthesizing conjugated trienoic acid in Intvitro which process comprises:

a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil,

b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about 78 C,

c. washing the acetone-insoluble residue with acetone at a temperature of about 78 C and then with ether,

d. removing the ether from (c) and drying the powder in vacuo,

e. triturating the powder from (d) in 0.1 M tris-HCl buffer,

f. filtering the suspension from (e) through glass wool,

g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes,

h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer,

. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 11 umoles of oxidized nicotinamide-adenine dinucleotide, about 1 1.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5'-diphosphate and adenosine-S'-triphosphate, about 1.7 mM MgSO,, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOl-l to neutralize the mixture,

j. incubating the reaction mixture from (i) at 30 C for a vitro which process comprises:

macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about 78 C,

. washing the acetone-insoluble residue with acetone at a temperature of about 78 C and then with ether,

. removing the ether from (c) and drying the powder in vacuo, triturating the powder from (d) in 0.1 M tris-HCl buffer, filtering the suspension from (e) through glass wool,

centrifuging the filtrate from (f) at about 1,100 g for about minutes,

. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer,

. homogenizing about 1 part of the enzyme from (h) with incubating the reaction mixture from (i) at 30 C for a period of about 45 minutes, isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.

A process for synthesizing conjugated trienoic acid in vitro which process comprises:

macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about -78 C,

washing the acetone-insoluble residue with acetone at a temperature of about 78 C and then with ether,

removing the ether from (c) and drying the powder in vacuo,

triturating the powder from (d) in 0.1 M tris-HCl buffer, filtering the suspension from (e) through glass wool,

centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes,

adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCI buffer,

. homogenizing about 1 part of the enzyme from (h) with incubating the reaction mixture from (i) at 30 C for a period of about 45 minutes,

isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane. 

2. A process for synthesizing conjugated trienoic acid in vitro which process comprises: a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about -78* C, c. washing the acetone-insoluble residue with acetone at a temperature of about -78* C and then with ether, d. removing the ether from (c) and drying the powder in vacuo, e. triturating the powder from (d) in 0.1 M tris-HCl buffer, f. filtering the suspension from (e) through glass wool, g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes, h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer, i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 15.7 mM of reduced nicotinamide-adenine dinucleotide about 11.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5''-diphosphate and adenosine-5''-triphosphate, about 1.7 mM MgSO4, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOH to neutralize the mixture, j. incubating the reaction mixture from (i) at 30* C for a period of about 45 minutes, k. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.
 3. A process for synthesizing conjugatEd trienoic acid in vitro which process comprises: a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about -78* C, c. washing the acetone-insoluble residue with acetone at a temperature of about -78* C and then with ether, d. removing the ether from (c) and drying the powder in vacuo, e. triturating the powder from (d) in 0.1 M tris-HCl buffer, f. filtering the suspension from (e) through glass wool, g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes, h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer, i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 15.7 mM of reduced nicotinamide-adenine dinucleotide, about 11.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5'' -diphosphate and adenosine-5''-triphosphate, about 1.7 mM MgSO4, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOH to neutralize the mixture, j. incubating the reaction mixture from (i) at 30* C for a period of about 45 minutes, k. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.
 4. A process for synthesizing conjugated trienoic acid in vitro which process comprises: a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about -78* C, c. washing the acetone-insoluble residue with acetone at a temperature of about -78* C and then with ether, d. removing the ether from (c) and drying the powder in vacuo, e. triturating the powder from (d) in 0.1 M tris-HCl buffer, f. filtering the suspension from (e) through glass wool, g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes, h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer, i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 11.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5''-diphosphate and adenosine-5''-triphosphate, about 1.7 mM MgSO4, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOH to neutralize the mixture, j. incubating the reaction mixture from (i) at 30* C for a period of about 45 minutes, k. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.
 5. A process for synthesizing conjugated trienoic acid in vitro which process comprises: a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about -78* C, c. washing the acetone-insoluble residue with acetone at a temperature of about -78* C and then with ether, d. removing the ether from (c) and drying the powder in vacuo, e. triturating the powder from (d) in 0.1 M tris-HCl buffer, f. filtering the suspension from (e) through glass wool, g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes, h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer, i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 11 Mu moles of oxidized nicotinamide-adenine dinucleotide, about 11.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5''-diphosphate and adenosine-5''-triphosphate, about 1.7 mM MgSO4, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOH to neutralize the mixture, j. incubating the reaction mixture from (i) at 30* C for a period of about 45 minutes, (k) isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.
 6. A process for synthesizing conjugated trienoic acid in vitro which process comprises: a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about -78* C, c. washing the acetone-insoluble residue with acetone at a temperature of about -78* C and then with ether, d. removing the ether from (c) and drying the powder in vacuo, e. triturating the powder from (d) in 0.1 M tris-HCl buffer, f. filtering the suspension from (e) through glass wool, g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes, h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer, i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 11 Mu moles of oxidized nicotinamide-adenine dinucleotide phosphate, about 11.4 mM of a nucleotide pyrophosphate selected from the group consisting of adenosine-5''-diphosphate and adenosine-5''-triphosphate, about 1.7 mM MgSO4, about 330 mM castor fatty acid, and a sufficient amount of 0.5 N KOH to neutralize the mixture, j. incubating the reaction mixture from (i) at 30* C for a period of about 45 minutes, k. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane.
 7. A process for synthesizing conjugated trienoic acid in vitro which process comprises: a. macerating in liquid nitrogen thinly sliced tissue from tung nuts which are actively accumulating oil, b. homogenizing the macerated tissue from (a) with acetone and sand at a temperature of about -78* C, c. washing the acetone-insoluble residue with acetone at a temperature of about -78* C and then with ether, d. removing the ether from (c) and drying the powder in vacuo, e. triturating the powder from (d) in 0.1 M tris-HCl buffer, f. filtering the suspension from (e) through glass wool, g. centrifuging the filtrate from (f) at about 1,100 g for about 10 minutes, h. adjusting the amount of enzyme in the supernatant from (g) to a concentration of about 15 to 35 mg of protein per ml by the addition of 0.1 M tris-HCl buffer, i. homogenizing about 1 part of the enzyme from (h) with about 0.43 to 0.66 parts of a mixture consisting of about 7.1 mM reduced coenzyme A, about 15.7 mM of reduced nicotinamide-adenine dinucleotide, about 1.7 mM MgSO4, about 330 mM castor fatty acid and a sufficient amount of 0.5 N KOH to neutralize the mixture, j. incubating the reaction mixture from (i) at 30* C for a period of about 45 minutes, k. isolating the conjugated trienoic fatty acid from the aqueous reaction mixture from (j) by adding about 1.33 parts of 0.5 N HCl and then about 10 parts of hexane. 